LNBio Mas Lab - PaesLeme_SalivaPeptidomics

Combined Peptidomic and Proteomic analysis of OSCC Saliva

  • Organism: Homo sapiens
  • Instrument: Xevo TQ-XS,ACQUITY UPLC
  • SpikeIn: Yes
  • Keywords: saliva, oral cancer, peptidome, proteolysis, protease inhibitors, oral squamous cell carcinoma
  • Lab head: Adriana Paes Leme
Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging since changes can occur simultaneously at protease, their inhibitor and substrate levels. In this work we combined peptidomics, proteomics and peptidase predictions for studying proteolytic events in saliva associated with oral squamous cell carcinoma (OSCC) prognosis. Our results suggest that cleavage products differentially abundant in the saliva of patients with (N+) or without (N0) nodal metastasis exhibit potential of prognostic value in oral cancer and were are able to discriminate N+ and N0 patients whereas reduced protein levels of peptidase inhibitors might disturb the proteolytic balance in saliva of OSCC patients with poor prognosis
Experiment Description
One µg of saliva digest was resolved over a 60-min gradient using an Acquity UPLC-Class M equipped with a trap column (Waters Acquity UPLC BEH C18 130A, 5 µm, 300 µm × 50 mm) and a BEH Shield C18 IonKey column (10 cm × 150 µm ID packed with 1.7 µm C18 particles, Waters, USA). Chromatographic conditions were as follows: flow rate at 1.2 µL/min, temperature set to 40°C, and acetonitrile (MeCN) gradient starting at 2% B (MeCN, 0.1% formic acid), following a linear ramp to 40% B over 45 min, followed by a step increase to 85% B until 47 min and conditioning at 2% B until 60 min. Mass spectrometry analysis of eluting peptides was performed via Selected-Reaction Monitoring (SRM-MS) using a Xevo TQ-XS Triple quadrupole coupled to the LC system. Quadrupoles Q1 and Q3 were operating as unit mass resolution (0.7 Th FWMH). Schedule SRM acquisition was adjusted to a 3-min elution window, with dwell times automatically set in MassLynx v4.2 to achieve at least 10 points per peak. The optimal collision energy was determined for each peptide by Skyline.
Sample Description
Saliva of OSCC patients with (N+) or without (N0) lymph node metastasis were centrifuged for 5 min at 1,500 × g, 4 °C to pellet intact cells and debris. Protein concentration was determined on the supernatant using Bradford assay kit (Bio-Rad). An aliquot with 10 µg of total protein was digested in solution using trypsin. In brief, samples were treated with urea buffer (100 mM Tris-HCl pH 7.5, 8 M urea, 2 M thiourea, 5 mM EDTA, 1 mM PMSF, and 1 mM DTT) containing cOmplete Mini Protease Inhibitor Cocktail (Roche) and the mixture sonicated in an ultrasound bath for 10 min. Following a centrifugation at 10,000 × g for 5 min, the supernatants were sequentially treated with 5 mM DTT (for 25 min, at 56 °C) and 14 mM iodoacetamide (for 30 min in the dark, at room temperature) for protein reduction and alkylation of cysteines. The mixture was then diluted with 50 mM ammonium bicarbonate to lower urea concentration to 1.6M, and calcium chloride added to 1mM final concentration. Samples were digested for 16h at 37 °C using sequencing grade modified trypsin (Promega) at 1:50 - enzyme to protein – ratio. The reaction was terminated by acidification with TFA to 0.4%. Peptides were desalted with StageTips C18 (3M Empore), dried in a vacuum concentrator and reconstituted in 0.1% formic acid. Stable isotope labeled peptides were spiked in the samples for relative quantification, and Pierce iRT mixture used to asses instrument performance.
Created on 7/7/20, 7:39 AM
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20200702_SRM_assay_2020-07-02_20-38-07.sky.zip2020-07-07 07:39:123111442119